1. Field of the Invention
The present invention relates generally to sensors that are used to detect and measure the presence of metal ions in solutions. More particularly, the present invention is directed to metal chelating lipids which are useful as sensors in fluorometric methods for detecting metal ions.
2. Description of Related Art
The detection of metal ions is an important area of analytical chemistry. A multitude of procedures and instruments is available for measuring a wide variety of metal ions, including copper, iron, lead, cadmium, mercury, nickel, cobalt, calcium and potassium. Many of these methods, however, depend upon highly specialized sensors to provide adequate levels of specificity and sensitivity. For example, flow injection atomic absorption spectrometry and size exclusion chromatography followed by inductively coupled plasma mass spectrometry (SEC/ICP-MS) are capable of detecting minute quantities of a wide variety of metal ions. However, such detection systems are very expensive to build and operate. In addition, the instrumentation is often very large and delicate and thus is restricted to use in a controlled laboratory environment. Other commonly used sensors, such as ion-selective electrodes (ISE), ion-selective field effect transistors (ISFET) and chemically modified field effect transistors (CHEMFET), are more convenient to use but are usually specific for a single metal ion, often lack high sensitivity, and require complex schemes for their manufacture.
Fluorimetry is an analytical method which has been used both to quantitatively and qualitatively detect metal ions. Fluorimetry is based on the ability of various molecular species (fluorophores) to absorb a photon of a particular energy (wavelength) and subsequently emit a second photon of lower energy (longer wavelength). The wavelength and intensity of the emitted light are often highly dependent on the chemical environment surrounding the fluorophore. Changes in the fluorescence emission properties of the fluorophore due to the presence of metal ions can be used to provide a measure of the ion concentration. An advantage of fluorimetric detection methods is that they are, in general, extremely sensitive.
An example of a fluorimetric sensing technique is fluorescent photoinduced electron transfer [PET; see Bissell et al., "Fluorescent PET (Photoinduced Electron Transfer) Sensors," Topics in Current Chemistry, 168 (1993), pp. 223-264]. In such a sensor, the detector molecule consists of a fluorophore that is linked by a spacer to an ion-binding receptor. Upon absorption of a photon the detector molecule can release the energy via either fluorescence or electron transfer. Binding of ions by the receptor perturbs the competition between these two processes and thus affects the intensity of light emitted.
Fluorescently-labeled synthetic lipids have been investigated recently for use in a variety of metal ion sensor systems, Lipids possess the ability to self-assemble into different structures (e.g. vesicle bilayers, monolayers, Langmuir-Blodgett (LB) multilayers, etc.), and thus are convenient starting materials for construction of a sensor. An exemplary lipid-based sensor for the detection of potassium ions is disclosed in an article written by Schaffer et al. entitled "Optical Sensors-Part 23. Effect of Langmuir-Blodgett Layer Composition on the Response of Ion-Selective Optrodes for Potassium, Based on the Fluorimetric Measurement of Membrane Potential" (Analyst, May 1989, Vol. 113, pp. 693-697). This sensor consists of a composite four layer LB film deposited onto a glass support. The bottom two layers, nearest the glass surface, consist of the neutral ion-carrier peptide, valinomycin, and an inert matrix fatty acid, arachidic acid. The top two layers, exposed to the surrounding medium, are composed of a lipid-linked fluorescent dye, rhodamine B, and arachidic acid. Valinomycin serves to concentrate the potassium (K.sup.+) ions near the lipid-water interface. The emission intensity of rhodamine in the lipid layer is strongly dependent upon the membrane potential between an aqueous sample solution and the lipid phase. The potential is in turn dependent upon the potassium concentration in the sample solution. Thus, the fluorescence intensity was found to decrease with increasing K.sup.+ concentration. The change in intensity was found to be linear with K.sup.+ concentration over the range 0.01-100 mM, and the selectivity factor for K.sup.+ over Na.sup.+, a common interfering ion, was 10.sup.4.
A second example of an ion sensor based on lipid LB films was described by W. Budach, et al. ("Metal Ion Sensor Based on Dioctadecyl-dithiocarbamate-Metal Complex Induced Energy Transfer," Thin Solid Films 210/211 (1992), pp. 434-436). This sensor also consists of four lipid layers on a glass support as follows: a hydrophobic anchor layer of eicosyl amine; a mixed layer of arachidic acid, arachidic methyl ester, and the fluorophore, N,N'-dioctadecyl-oxacyanine perchlorate (S9); a spacer layer of arachidic acid; and the metal ion-binding layer, dioctadecyldithio-carbamate (DOTC), which is exposed to the aqueous solution. The fluorescence emission spectrum of the dye shows significant ovarlap with the absorption spectrum of the Cu-DOTC complex. Thus, in the presence of Cu.sup.2+ some fraction of the excited dye molecules will relax to the ground state via energy transfer to the Cu-DOTC complex rather than via fluorescence, and a decrease in the emission intensity is observed. This sensor was incorporated into a flow cell and found to have a sub-micromolar detection limit for Cu.sup.2+ and a response time of only a few seconds.
While LB films have the advantage of forming well-ordered, two-dimensional architectures which are highly desired in many sensors, they suffer from a lack of stability and are difficult to produce in large quantities. Lipid bilayer vesicles, in contrast, are relatively simple to prepare, even in large quantities, and thus are an attractive framework on which to build a sensing device. Like LB films, lipid bilayer vesicles are spontaneously assembled from their lipid components to yield a degree of organization not easily accessible with other types of molecules. An example of a metal ion sensor based on lipid vesicles and UV absorbance (not fluorimetry) has been described by Shimomura and Kunitake ("Interaction of Ions with the Surface Receptor of the Azobenzene-Containing Bilayer Membrane. Discrimination, Transduction, and Amplification of Chemical Signals," J. Am. Chem. Soc. 104 (1982), pp. 1757-1759). This simple system consists of two lipids, an azobenzene-containing dye lipid with a metal-binding ethylenediamine headgroup and a matrix lipid, dioctadecyl dimethylammonium bromide (DODAB), co-sonicated to form mixed-lipid bilayer vesicles. At low pH the dye lipid headgroup is positively charged and the lipid is dispersed throughout the bilayer exhibiting an absorbance maximum at 355 nm. When a divalent anion such as SO.sub.4.sup.2- is added, the anion can bridge the positively charged headgroups of the dye lipid causing aggregation of the dye and a concomitant hypsochromic shift of the absorbance to 312 nm. If the same dye is mixed with a slightly different matrix lipid which inhibits aggregation of the dye lipid at neutral pH (where the headgroup is unprotonated), the monomer absorbance is again observed at 355 nm. When Cu.sup.2+ is added to the solution, chelation of the metal ion by the dye headgroup results in a new absorption maximum of the azobenzene cluster at 312 nm.
Recently, an improvement on this system has bee; reported by Singh, et al. ("Metal Ion Induced Phase Changes in Self-Assembled Membranes," Langmuir8 (1992), pp. 1570-1577) in which the amine head group of the azobenzene dye was replaced with cyclam, a strong chelator for Cu.sup.2+. This cyclam azobenzene amphiphile (CABA) and a similar ammonium bromide matrix lipid (1:30 molar ratio) were formed into mixed bilayer vesicles. CABA was found to be dispersed as the isolated species within the fluid matrix above 24.5.degree. C., with an absorbance maximum at 360 nm. At temperatures in the range 24.5.degree.-45.degree. C., chelation of Cu.sup.2+ by the cyclam headgroup results in formation of azobenzene dye clusters, which results in a shift in the absorbance from 360 to 320 nm. A linear increase in the absorbance of vesicles at 326 nm with increasing Cu.sup.2+ concentration in the range of 6-36 .mu.M was observed.
Although the azobenzene lipid system described above is intriguing due to its simplicity (only two component molecules) and the ease with which it can be produced, several problems remain which make this system unsuitable for production of a practical sensor. First, temperature cycling in the range of 10.degree. to 80.degree. C. was required to allow the necessary phase change to occur. Secondly, the UV wavelength monitored, 326 nm, is well out of the visible spectrum and therefore requires the use of a spectrophotometer and quartz optics. Finally, the restricted range of concentrations measurable (less than an order of magnitude) and the relatively low sensitivity greatly limit its application.
Although a great deal of progress has been made in investigating possible modified lipids for use as detectors, there is still a continuing need to further investigate modified lipids in order to provide improved detectors which are easy to use and have the desirable characteristics mentioned above.